**Aflatoxin Measurement and Analysis**

#### Peiwu Li and Qi Zhang et al.\*

*Key Laboratory of Biotoxin Analysis of Ministry of Agriculture, Key Laboratory of Oil Crops Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, China* 

#### **1. Introduction**

182 Aflatoxins – Detection, Measurement and Control

Zhang, J.; Song, S.; Wang, L.; Pan, D.& Fan, C. (2007). A gold nanoparticle-based

Zhao, G.; Feng, J.J.; Zhang, Q.L.; Li, S.P. & Chen, H.Y. (2005).Synthesis and Characterization

pp.2888-2893,

chronocoulometric DNA sensor for amplified detection of DNA, *Nat. Protoc.*, Vol.2,

of Prussian Blue Modified Magnetite Nanoparticles and Its Application to the Electrocatalytic Reduction of H2O2, *Chem. Mater.*, Vol.17, No.12, pp. 3154–3159

> Aflatoxin is a group of secondary metabolites produced by fungi *Aspergillus* species, such as *A. flavus* and *A. parasiticus;* in particular, *A. flavus* is common in agriculture. *A. bombycis*, *A. ochraceoroseus*, *A. nomius*, and *A. pseudotamari* are also aflatoxin-producing species, but they are encountered much less frequently (Bennett and Klich, 2003).

> Aflatoxin contamination can be occurred very widely. They can be found in over a hundred kinds of agro-products and foods,such as peanut, corn, rice, soy sauce, vinegar, plant oil, pistachio, tea, Chinese medicinal herb, egg, milk, feed etc,. Also some of them in animal organism can be detected. Besides these, aflatoxin can spread and accumulated in environment, for example, river and agricultural field.

> Aflatoxins are highly toxic, mutagenic, teratogenic, and carcinogenic compounds, a group of difuranocoumarin derivatives, consisted of a coumarin and a double-furan-ring of molecule usually. Aflatoxin B1, for example, its toxicity is ten times of potassium cyanide, 68 times of arsenic and 416 times of melamine. Furthermore, their carcinogenicity is over 70 times than that of dimethylnitrosamine and 10000 times of Benzene Hexachloride (BHC). And International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) accepted that aflatoxin should be classified as a Group 1 carcinogen in 1987, and then AFB1 is classified as Group 1 (carcinogenic to humans) by the WHO– IARC in 1993 (Li, Zhang & Zhang, 2009). According to the nearest researches by University of Pittsburgh, aflatoxin may play a causative role in 4.6–28.2% of all global HCC cases (Liu and Wu, 2010).

> To protect agricultural environment, estimate quality of commercials of agro-products and food, and safeguard safety of consumers' health and lives, over seventy countries setup maximum limits in agro-products, and analytical methods for determination of aflatoxin, play a great role for monitoring and estimation of the contaminants.

> There are a variety of well established methodologies reported for analysing aflatoxins in many different foodstuffs, such as thin layer chromatography, high-performance liquid chromatography, ultra-pressured layer chromatography, immunoaffinity chromatographyhigh-performance liquid chromatography, near infrared spectroscopy and immunoassay

<sup>\*</sup> Daohong Zhang, Di Guan, Xiaoxia, Ding Xuefen Liu, Sufang Fang, Xiupin Wang and Wen Zhang *Key Laboratory of Biotoxin Analysis of Ministry of Agriculture, Key Laboratory of Oil Crops Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, China.*

Aflatoxin Measurement and Analysis 185

Elution. By passing a solvent such as acetonitrile through the IAC, breaking the antibodyaflatoxin bond, the captured aflatoxin is removed from the antibody and thus eluted from the column. The big volume of sample loading and the small volume of solvent eluting make the analyte concentrate. The eluate containing aflatoxin is then further developed by

The principle of solid phase extraction (SPE) columns is a variation of chromatographic techniques that uses a solid phase and a liquid phase to isolate one, or one type, of analyte from a solution. The columns contain different packing materials, ranging from silica gel, C-18 (octadecylsilane), florisil, phenyl, aminopropyl, ion exchange materials, both anionic and cationic, and molecular imprinted polymers (Giraudi et al, 2007; Jornet et al, 2000; Mateo et al, 2002; Vatinno et al, 2008; Yu & Lai, 2010; Zambonin et al, 2001). The generally procedure is to load the sample into column, retain the analyte, wash away impurities, and then elute the analyte. A MycoSep multifunctional cleanup column has been developed for one step clean-up of aflatoxin (Figure 2). The MycoSep clean-up column is pushed into a test tube (containing the sample), forcing the sample to filter upwards through the packing material of the column. The interferences adhere to the chemical packing in the column and the purified extract, containing the aflatoxin of interest, passes through a membrane (frit) to the surface of the column. The method is rapid, simple and economical due to the fact that the clean-up of aflatoxin from the column is a single pass procedure using the extract solvent as the eluting solvent. The column has a long shelf-life because it contains no biological reagents, and can be stored at room temperature. However, unlike immunoaffinity columns, the MycoSep clean-up column cannot concentrate the analyte during the clean-up procedure, and also the recovery may vary depending upon the complexity of the food

Fig. 2. Scheme of aflatoxin multifunctional cleanup column for sample pretreatment

addition of fluorescence enhancer or directly measured by HPLC method.

samples (Zheng et al, 2006).

(clean-up).

methods. We here will not only demonstrate current such analytical methods for aflatoxins, but also illuminate tomorrow's trends on analysis of aflatoxins. To help readers understand them well, some basic information of these methods were also presented, including principle of developing, choosing and using these methods.

#### **2. Pretreatment of sample**

#### **2.1 Immunoaffinity or multipurification column**

The immunoaffinity column (IAC) occupies a special place among the immune analytical approaches, being used many years as a method of sample purification and concentration in the aflatoxin analysis (Scott & Trucksess, 1997). The principle of the IAC is that an antibody (polyclonal or monoclonal) recognized the analyte is immobilized onto a solid support such as agarose or silica in phosphate buffer, all of which is contained in a small column.

The clean-up procedures are completed in four steps (Figure 1):

Condition. The column is initially conditioned with phosphate buffered saline (PBS) and reaches room temperature.

Loading of the sample. The crude sample extract is applied to the IAC containing specific antibodies to aflatoxin at slow steady flow rate of 2-3 mL/min. Gravity or vacuum system can be used to control flow rate. The aflatoxin binds to the antibody and is retained in the IAC. The crude sample extract must be in aqueous solution because organic solvents can damage the antibody and can interfere with the antibody-aflatoxin interaction. The binding strength of the antibody-aflatoxin will influence recovery of the IAC. The specificity of antibody is important to remove the structurally closely compounds which can cause interferences in the quantitation of aflatoxin. The capacity of the IAC (the total number of antibody sites available for binding aflatoxin) is also important as overloading the column will lead to poor recovery (Senyuva & Gilbert, 2010).

Washing. The column is washed with washing solution (water or phosphate buffered saline) to remove impurities. After washing completely, the IAC is blown to dryness by N2 stream.

Fig. 1. Scheme of aflatoxin immunoaffinity column for sample pretreatment (clean-up and enrichment).

methods. We here will not only demonstrate current such analytical methods for aflatoxins, but also illuminate tomorrow's trends on analysis of aflatoxins. To help readers understand them well, some basic information of these methods were also presented, including

The immunoaffinity column (IAC) occupies a special place among the immune analytical approaches, being used many years as a method of sample purification and concentration in the aflatoxin analysis (Scott & Trucksess, 1997). The principle of the IAC is that an antibody (polyclonal or monoclonal) recognized the analyte is immobilized onto a solid support such

Condition. The column is initially conditioned with phosphate buffered saline (PBS) and

Loading of the sample. The crude sample extract is applied to the IAC containing specific antibodies to aflatoxin at slow steady flow rate of 2-3 mL/min. Gravity or vacuum system can be used to control flow rate. The aflatoxin binds to the antibody and is retained in the IAC. The crude sample extract must be in aqueous solution because organic solvents can damage the antibody and can interfere with the antibody-aflatoxin interaction. The binding strength of the antibody-aflatoxin will influence recovery of the IAC. The specificity of antibody is important to remove the structurally closely compounds which can cause interferences in the quantitation of aflatoxin. The capacity of the IAC (the total number of antibody sites available for binding aflatoxin) is also important as overloading the column

Washing. The column is washed with washing solution (water or phosphate buffered saline) to remove impurities. After washing completely, the IAC is blown to dryness by N2 stream.

Fig. 1. Scheme of aflatoxin immunoaffinity column for sample pretreatment (clean-up and

as agarose or silica in phosphate buffer, all of which is contained in a small column.

principle of developing, choosing and using these methods.

The clean-up procedures are completed in four steps (Figure 1):

**2.1 Immunoaffinity or multipurification column** 

will lead to poor recovery (Senyuva & Gilbert, 2010).

**2. Pretreatment of sample** 

reaches room temperature.

enrichment).

Elution. By passing a solvent such as acetonitrile through the IAC, breaking the antibodyaflatoxin bond, the captured aflatoxin is removed from the antibody and thus eluted from the column. The big volume of sample loading and the small volume of solvent eluting make the analyte concentrate. The eluate containing aflatoxin is then further developed by addition of fluorescence enhancer or directly measured by HPLC method.

The principle of solid phase extraction (SPE) columns is a variation of chromatographic techniques that uses a solid phase and a liquid phase to isolate one, or one type, of analyte from a solution. The columns contain different packing materials, ranging from silica gel, C-18 (octadecylsilane), florisil, phenyl, aminopropyl, ion exchange materials, both anionic and cationic, and molecular imprinted polymers (Giraudi et al, 2007; Jornet et al, 2000; Mateo et al, 2002; Vatinno et al, 2008; Yu & Lai, 2010; Zambonin et al, 2001). The generally procedure is to load the sample into column, retain the analyte, wash away impurities, and then elute the analyte. A MycoSep multifunctional cleanup column has been developed for one step clean-up of aflatoxin (Figure 2). The MycoSep clean-up column is pushed into a test tube (containing the sample), forcing the sample to filter upwards through the packing material of the column. The interferences adhere to the chemical packing in the column and the purified extract, containing the aflatoxin of interest, passes through a membrane (frit) to the surface of the column. The method is rapid, simple and economical due to the fact that the clean-up of aflatoxin from the column is a single pass procedure using the extract solvent as the eluting solvent. The column has a long shelf-life because it contains no biological reagents, and can be stored at room temperature. However, unlike immunoaffinity columns, the MycoSep clean-up column cannot concentrate the analyte during the clean-up procedure, and also the recovery may vary depending upon the complexity of the food samples (Zheng et al, 2006).

Fig. 2. Scheme of aflatoxin multifunctional cleanup column for sample pretreatment (clean-up).

Aflatoxin Measurement and Analysis 187

extraction (SPE-CN) or immunoaffinity column (IAC) combined with application of

Chromatography columns were the most important part of the HPLC, normal and reversedphase columns were used for separation and purification of toxins depending on their polarity. Reversed-phase C18 columns with methanol–water or acetonitrile–water mobile

Modern analysis of mycotoxins relies heavily on HPLC employing various adsorbents depending on the physical and chemical structure of the mycotoxins. The use of the HPLC in determination of aflatoxins and their metabolites showed higher levels of accuracy and lower detection limits when using SPE-CN or IAC regardless of the HPLC detectors used. Zhao used UPLC for determinations of Aflatoxins B1, B2, G1 and G2 (AFB1, AFB2, AFG1 and AFG2), and the detection limits (S/N = 3) for B1, B2, G1 and G2 were 0.32, 0.19, 0.32 and 0.19μg kg-1, the corresponding quantification limits (S/N = 10) were 1.07, 0.63, 1.07 and

Derivative with a suitable fluorophore can enhance the natural fluorescence of aflatoxins,which can improve the fluorescence detection sensitivity. The present needs for HPLC fluorescence detection of aflatoxins determination in food and feedstuffs are an emphasis on the improvement of the sampling and extraction steps to lead to more accurate determinations, and further investigations of non-destructive pre-column or post-column derivative methods appears to be a large unexplored field. Some aflatoxins like aflatoxin B1, aflatoxin G1, because of its low signal or its easy quenching signals, several derivation

There are mainly three kinds of derivatizations: TFA, halogen, and its derivatives, metal ions (Hg2+), cyclodextrine and its derivatizations. The enhancement mechanisms varies

AFB1 derivative method is mainly based on hydrolysis of the second furan ring in acidic solution, and AFB1 is transformed into B2a ,which makes a fluorescent greatly enhanced.This mechanism is commonly used by TFA,halogen,and its derivatives (PBPB)

Dr. Ma (2007) had studied on the metal ions (Hg2+) enhancement for aflatoxins and proposed the probably mechanism was that AFB1 can be chelated with Hg2+, the propose of the complexes fluorescence can be enhanced, the speculate metal complexes electronic transition occurred ligand AFB1 to employed by Hg (II), the charge transfer transition metal ions, namely ligand-to-metal charge transition (LMCT) transition. LMCT transition with high energy, and its absorb is in the UV area, LMCT transition is occurred against bonding σ orbital, electronic horizontally inspire with ligand AFB1 oxidation and reduction of metal, occurred by electron reaction. Metal ions are two ligand simultaneously electronic warp reduction. Speculation that ligand AFB1 is probably in the form of ·L base separation formed 2·L or formed new molecular L - L or L - M2+ -L, reactant system rigid structure to strengthen or conjugated system increased, fluorescent intensity was greatly enhanced

The main reaction procedure may be described by the next response equations:

<sup>2</sup> 2 *n n LM L M L*

FLD(Brera et al, 2007; Edinboro, & Karnes, 2005; Jaimez & Fente, 2000)

phases, is most commonly used for aflatoxins in most laboratories.

0.63μg kg-1, respectively (Fu et al, 2008).

**Fluorescence enhancement methods of aflatoxins** 

reagents were used during the detection procedure.

etc.(Francis et al., 1988; Joshua, 1993; Braga et al, 2005)

with different kinds of derivatizations.

(Ma, 2007).

#### **2.2 How to simplify current protocol**

The selection of pretreatment methods for samples depends mainly on two aspects: one is the analytical methods adopted, another is samples to be analyzed. The former is more importent with great differences according to the kinds of analytical methods. Complexity, time consuming and cost are the main factors contributed the popular degree by operators and practicability in on-site use. Among these factors complexity degree is most concerned for the exposure hazards of aflatoxins.

Sample pretreatment for instrumental analysis (e.g., HPLC, GC, LC/MS and GC/MS) is very tedious, expensive and time consuming, and needs well equipped laboratories to accomplish it, e.g., frequently involving in large-scale equipment, large sample volumes, extensive extraction or derivatization steps (Tang et al., 2008), complicate clean-up and concentration, and multiple centrifugation, etc. While for immunoassay (for instance, enzyme-linked immunosorbent assay, ELISA) it is usually easier, cheap and rapid generally without derivation but still need clean-up and concentration. How to simplify current pretreatment protocol is a question to extend the methods for aflatoxins detecting outside the laboratory. As an alternative, lateral-flow immunochromatographic assay combines chromatography with immunoassay with less interference due to chromatographic separation, offers the advantages of most simple, cheap and time-saving, requiring only a simple extraction step (Tanaka et al., 2006) or even no need for extraction (e.g., detection of aflatoxin M1 in milk). Therefore, the pretreatment protocol of sample can be simplified by adopting suitable analytical methods, e.g., immunochromatographic assay.

### **3. Sample analysis**
